This is a resubmission of an R21 application. The goal we expect to reach is to label renewable neural stem cells in vitro and in vivo to identify the precise location of neural stem cells in the brain and their developmental fates, this knowledge is a prerequisite for developing safe clinical applications of neural stem cells for the purpose of treating neurodegenerative disorders and CNS injuries. We propose to use a stem cell specific, MELK gene regulatory element to drive the Fluorescent Timer reporter. Additional preliminary data provide convincing evidence of the specific expression of the MELK gene in two types of somatic stem cells, neural and hematopoietic. Critically, a 3.4kb genomic fragment upstream of the MELK coding region is selectively active in neurosphere-forming cells and in some Nestin positive cells but not in GFAP positive astrocytes or TuJ1 positive young neurons. For the reporter gene, we will take advantage of the Fluorescent Timer protein, a special variant of the red fluorescent protein that spontaneously changes its fluorescence color from green to red with maturation. Preliminary studies have shown that the Fluorescent Timer expressed under the control of a Nestin enhancer provides a clear fluorescent distinction among all neural precursors and terminally differentiated neural cells. The broad expression pattern of the Nestin enhancer, however, makes this combination unsuitable for the precise labeling of neural stem cells in vitro or in vivo. In summary, our current data strongly supports the hypothesis that the MELK-Timer combination will permit more precise labeling and the separation of neural stem cells in vitro and in vivo. Our specific Aims are: 1. To investigate if the MELK promoter - Fluorescent Timer combination labels neural stem cells in neurospheres and during in vitro differentiation of ES cells, and, 2. To genetically label neural stem cells in vivo using the MELK - Fluorescence Timer transgenic or knock-in strategies. The genetic 'labeling' of neural stem cells will provide the model system to permit definitive studies of the stem cell niche, a specialized microenvironment that controls their self-renewal and differentiation. It is likely that more than one type of neural stem cells exist in the mammalian brain. The genetic labeling approach will address this issue providing a clear marker for the genetically defined stem cell population, whose developmental fate could then be precisely addressed both in vitro and in vivo.